An immunoassay is an advantageous method for determining and analyzing an antibody substance or an antigenic substance in that it is simple and has high specificity.
In particular, an immune complex transfer immunoassay is known to suitably suppress adsorption of contaminant protein onto solid phase and thereby-caused occurrence of background signals, which are the defects of sandwich method, and enable high sensitivity assay of an antibody substance or an antigenic substance in a test solution.
As used herein, the antibody substance or antigenic substance to be assayed in a test solution is referred to as test substance.
An immune complex transfer immunoassay is an immunoassay comprising, for example, two cycles of trapping using a pair of solid phases. The operation thereof is schematically explained by referring to an assay of antibody, as an example, from among the test substances. For the explanation's sake, one solid phase from the pair of solid phases, which is used for the first trapping, is referred to as the first solid phase and the other solid phase used for the next trapping is referred to as the second solid phase.
The typical immune complex transfer immunoassay comprises the following steps.
(a) An antigen modified with a functional group and a labeled antigen are bound to a target antibody to form an immune complex having a structure of "functional group-antigen-antibody-antigen-label" as shown, for example, in FIG. 5(a) with a reference number 50.
(b) The immune complex is trapped on a first solid phase via a certain moiety (functional group bound to the antigen in this case) in the immune complex. The immune complex may be trapped after being formed in a test solution or may be completed with respect to one end (functional group) thereof trapped earlier on a solid phase.
(c) The immune complex is released from the first solid phase in a solution used for transferring the immune complex.
(d) The immune complex is trapped on a second solid phase. The moiety of the immune complex which is used for the trapping is different from the moiety used in (b).
(e) The target antibody in the immune complex is assayed using the label conjugated in (a).
More detailed explanation and working examples of immune complex transfer immunoassay are found in the following literature.
(1) ISHIKAWA, E., HASHIDA, S., KOHNO, T., Development of ultrasensitive enzyme immunoassay reviewed with emphasis on factors which limit the sensitivity, MOLECULAR AND CELLULAR PROBES, 5, pp 81-95 (1991) PA1 (2) ISHIKAWA, E., HASHIDA, S., KOHNO, T., et al., Principle and applications of ultrasensitive enzyme immunoassay (Immune complex transfer enzyme immunoassay) for antibodies in body fluids, J. CLIN. LABORATORY ANALYSIS, 7, pp 376-393 (1993) PA1 (3) U.S. Pat. No. 5,236,849 and EP 303229 entitled Method of high sensitivity immunoassay PA1 (4) U.S. Pat. No. 5,236,830 and EP 368273 entitled Novel method for assaying antigen PA1 (5) ISHIKAWA, Eiji, Ultrasensitive enzyme immunoassay, Gakkai Shuppan Center (1993) PA1 (a) a step comprising coating an antibody on a dip stick type solid phase, and dipping the dip stick type solid phase in a test solution in a well type solid phase to trap the assay target, and PA1 (b) a step comprising rinsing the dip stick type solid phase, dipping same in a marker solution placed in a different well type solid phase and assaying the assay target. PA1 (a) coating a sustained release marker dissolved in a sucrose solution and the like on a well type solid phase, PA1 (b) placing a test solution in the well type solid phase, and PA1 (c) dipping an antigen-bound dip stick type solid phase in said test solution, thereby trapping the assay target antibody and simultaneously binding the marker. PA1 (A): a substance having a reactive group which specifically binds to a functional group previously introduced onto a substance which specifically forms an immune complex with a test substance PA1 (B): a substance having a reactive group capable of specifically binding to the test substance in the immune complex, a substance which specifically forms an immune complex with the test substance, or a functional group previously introduced onto said substance, provided that the moiety which binds to the reactive group of (A) does not bind to the reactive group of (B) and vice versa. PA1 (C): at least three positions determined on the periphery of the body of the dip stick type solid phase, wherein the distance between the positions is that which prevents the body of the dip stick type solid phase from contacting the well type solid phase when protrusions are formed at all of these positions.
In the conventional immune complex transfer immunoassay, plastic balls called beads having a diameter of about 3 mm are used as the first and the second solid phases. The beads are stirred in a test tube to perform the above-mentioned immune complex transfer immunoassay comprising the steps (a) to (e).
This method requires each one of the beads to be removed from and placed into test tubes with tweezers upon visually discriminating the two kinds of beads, thus raising problems in terms of handling easiness and the possibility of contamination of test tubes caused by carrying immune complex between test tubes with tweezers.
In addition, the possibility of a trace amount of immune complex released from the first solid phase contacting the second solid phase is very low, and only when a substance coated on the second solid phase has high affinity for the binding site of the immune complex, sufficient trapping is accomplished, which in turn results in a tendency that the amount of signals becomes less.
For conventional immunoassay, there have been known assay plates wherein a dip stick type solid phase and a well type solid phase are combined.
U.S. Pat. No. 3,826,619 describes:
The dip stick type solid phase used in these steps functions merely as a means for trapping the assay target, and the well type solid phase is nothing but a container to keep a solution.
Japanese Patent Application under WO 82/00058 teaches a special one step sandwich immunoassay using the above-mentioned assay plate wherein the dip stick type solid phase and the well type solid phase are combined, which comprises:
This method rather resembles the method using the immunoassay plate of the present invention in that it uses a well type solid phase and a dip stick type solid phase in combination. However, the well type solid phase only functions as a marker carrier, and to improve assay target-marking performance.
In contrast to the immunoassay using conventional plates, an immune complex transfer immunoassay comprises a specific process of transferring an immune complex containing a trace amount of an assay target from the first solid phase to the second solid phase, but an immunoassay apparatus capable of sufficiently accomplishing the transfer process has not been known.
Hence, there has been a demand for the development of an immunoassay apparatus capable of performing immune complex transfer immunoassay with ease and at high sensitivity, which can be suitably applied to clinical tests.